New cysE-pyrE-linked rfa mutation in Escherichia coli K-12 that results in a heptoseless lipopolysaccharide.

A new novobiocin-supersensitive mutant of Escherichia coli K-12 has been characterized biochemically and genetically. Lipopolysaccharide prepared from this mutant strain is truncated and contains 2-keto-3-deoxyoctulosonic acid as its only core sugar. This new core-defective mutation, designated rfa-2, results in increased sensitivity to several hydrophobic and some hydrophilic agents. Genetic analysis of the rfa mutant indicated that the rfa-2 locus is located at 81 min on the chromosome. The order of the genes in this region based on transduction analysis is xyl cysE rfa-2 rfaD70 pyrE. P1 transduction analyses indicate that the rfa-2 marker is nonallelic with the recently described cysE-pyrE-linked rfaD70 locus. Plasmids carrying the wild-type rfaD70+ allele failed to abolish the rfa-2 phenotypes. Further, the rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, was detected in crude extracts of a rfa-2 mutant strain, CL609, and was absent in the rfaD70 mutant. The wild-type rfa-2 allele codes either for a specific heptose biosynthetic enzyme (different from the rfaD gene product) or an enzymatic activity required for the addition of heptose to the lipid A-2-keto-3-deoxyoctulosonic acid acceptor.     

Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene.

The rfaD gene encodes ADP-L-glycero-D-mannoheptose-6-epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycerol-D-mannoheptose. The precise localization of the rfaD gene on a 1.3-kilobase SspI-HpaI fragment is reported. The rfaD gene and the flanking regions were completely sequenced. The location of the rfaD gene on the physical map of the Escherichia coli chromosome was determined. Primer extension studies were used to define the regulatory region of the rfaD gene. The cloned rfaD gene directed the synthesis of a 37,000-dalton polypeptide in several in vivo and in vitro expression systems. N-terminal analysis of purified ADP-L-glycero-D-mannoheptose-6-epimerase confirmed the first 34-amino-acid sequence deduced from the nucleotide sequence of the rfaD gene coding region. The primary structure of the rfaD protein contains the sequence fingerprint for the ADP-binding beta alpha beta fold at the N terminus.     

Does polymyxin B nonapeptide increase outer membrane permeability in antibiotic supersensitive enterobacterial mutants?

Abstract The outer-membrane-disorganizing peptide (polymyxin B nonapeptide; PMBN) was able to sensitize even "antibiotic supersensitive" enterobacterial mutants to hydrophobic antibiotics. This resulted in an extreme sensitivity. The mutants included the "deep rough" lipopolysaccharide mutants, as well as the acrA mutant of Escherichia coli and the "class A, B, and C mutants of Salmonella typhimurium . Sensitization factors of approx. 30 or more were found for most antibiotics. Even minimum inhibitory concentrations as low as approx. 0.5 ng/ml (rifampicin), 1.5 ng/ml (erythromycin), 2 ng/ml (fusidic acid), 6 ng/ml (novobiocin), and 30 ng/ml (clindamycin) were achieved in the presence of 30 μg/ml of PMBN. The finding indicates that the mechanisms which mediate the increase in hydrophobic diffusion are different but synergistic in the mutants and in the PMBN-grown cells.     

Altered phospholipid composition in mutants of Escherichia coli sensitive or resistant to organic solvents.

Mutants of Escherichia coli with altered resistance to low molecular weight organic solvents were isolated. Solvent-resistant mutants showed a decrease in the ratio of phosphatidylethanolamine to the anionic phospholipids (phosphatidylglycerol and cardiolipin) relative to the wild-type, whereas solvent-sensitive strains showed an increase. Reversion studies on representative mutants demonstrated that the phenotypic response to solvents and the changes in phospholipid composition were genetically associated. The fatty acid and lipopolysaccharide compositions of the various mutants showed no significant differences from the parental strain. The lesions in two of the solvent-sensitive mutants (DC7 and DC9) and one of the resistant mutants (DC11) were mapped by cotransduction with phage P1 and shown to lie very close to the pss locus at 56 min on the Escherichia coli map.     

Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides.

We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides. Four phenotypic classes were identified, including two with a novel array of characteristics. The mutations mapped to two genetic loci. Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides. Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene.     

New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the σ^E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA , that decreased the σ^E-dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM ( rfaD ) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM ( rfaD ) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn 10 insertions were isolated, which led to an increased activity of the σ^E regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA , fkpA and ompH/skp genes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.     

Chromosomal and plasmid-encoded enzymes are required for assembly of the R3-type core oligosaccharide in the lipopolysaccharide of Escherichia coli O157:H7

The type R3 core oligosaccharide predominates in the lipopolysaccharides from enterohemorrhagic Escherichia coli isolates including O157:H7. The R3 core biosynthesis (waa) genetic locus contains two genes, waaD and waaJ, that are predicted to encode glycosyltransferases involved in completion of the outer core. Through determination of the structures of the lipopolysaccharide core in precise mutants and biochemical analyses of enzyme activities, WaaJ was shown to be a UDP-glucose:(galactosyl) lipopolysaccharide alpha-1,2-glucosyltransferase, and WaaD was shown to be a UDP-glucose:(glucosyl)lipopolysaccharide alpha-1,2-glucosyltransferase. The residue added by WaaJ was identified as the ligation site for O polysaccharide, and this was confirmed by determination of the structure of the linkage region in serotype O157 lipopolysaccharide. The initial O157 repeat unit begins with an N-acetylgalactosamine residue in a beta-anomeric configuration, whereas the biological repeat unit for O157 contains alpha-linked N-acetylgalactosamine residues. With the characterization of WaaJ and WaaD, the activities of all of the enzymes encoded by the R3 waa locus are either known or predicted from homology data with a high level of confidence. However, when core oligosaccharide structure is considered, the origin of an additional alpha-1,3-linked N-acetylglucosamine residue in the outer core is unknown. The gene responsible for a nonstoichiometric alpha-1,7-linked N-acetylglucosamine substituent in the heptose (inner core) region was identified on the large virulence plasmids of E. coli O157 and Shigella flexneri serotype 2a. This is the first plasmid-encoded core oligosaccharide biosynthesis enzyme reported in E. coli.     

Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis.

A genetically defined mutation, designated rfa-2, results in altered lipopolysaccharide (LPS) biosynthesis. rfa-2 mutants produce a core-defective LPS that contains lipid A and a single sugar moiety, 2-keto-3-deoxyoctulosonic acid, in the LPS core region. Such LPS core-defective or deep-rough (R) mutant structures were previously designated chemotype Re. Phenotypically, rfa-2 mutants exhibit increased permeability to a number of hydrophilic and hydrophobic agents. By restriction analyses and complementation studies, we clearly defined the rfa-2 gene on a 1,056-bp AluI-DraI fragment. The rfa-2 gene and the flanking rfa locus regions were completely sequenced. Additionally, the location of the rfa-2 gene on the physical map of the Escherichia coli chromosome was determined. The rfa-2 gene encodes a 36,000-dalton polypeptide in an in vivo expression system. N-terminal analysis of the purified rfa-2 gene product confirmed the first 24 amino acid residues as deduced from the nucleotide sequence of the rfa-2 gene coding region. By interspecies complementation, a Salmonella typhimurium rfaC mutant (LPS chemotype Re) is transformed with the E. coli rfa-2+ gene, and the transformant is characterized by wild-type sensitivity to novobiocin (i.e., uninhibited growth at 600 micrograms of novobiocin per ml) and restoration of the ability to synthesize wild-type LPS structures. On the basis of the identity and significant similarity of the rfa-2 gene sequence and its product to the recently defined (D. M. Sirisena, K. A. Brozek, P. R. MacLachlan, K. E. Sanderson, and C. R. H. Raetz, J. Biol. Chem. 267:18874-18884, 1992), the S. typhimurium rfaC gene sequence and its product (heptosyltransferase 1), the E. coli K-12 rfa-2 locus will be designated rfaC.     

Genetic and molecular analyses of Escherichia coli K1 antigen genes

The plasmid pSR23, composed of a 34-kilobase E. coli chromosomal fragment inserted into the BamHI site of the pHC79 cosmid cloning vector, contains genes encoding biosynthesis of the K1 capsular polysaccharide. Deletions, subclones, and Tn5 insertion mutants were used to localize the K1 genes on pSR23. The only deletion derivative of pSR23 that retained the K1 phenotype lacked a 2.7-kilobase EcoRI fragment. Subclones containing HindIII and EcoRI fragments of pSR23 did not produce K1. Cells harboring pSR27, a subclone containing a 23-kilobase BamHI fragment, synthesized K1 that was not detectable extracellularly. Six acapsular Tn5 insertion mutants of three phenotypic classes were observed. Class I mutants synthesized K1 only when N-acetylneuraminic acid (NANA) was provided in the medium. Reduced amounts of K1 were detectable in cell extracts of class II mutants. Class III mutants did not produce detectable K1 in either extracts or when cells were provided exogenous NANA. All mutants had sialyltransferase activity. Analysis in the E. coli minicell system of proteins expressed by derivatives of pSR23 identified a minimum of 12 polypeptides, ranging in size from 18,000 to 80,000 daltons, involved in K1 biosynthesis. The 16-kilobase coding capacity required for the proteins was located in three gene clusters designated A, B, and C. We propose that the A cluster contains a NANA operon of two genes that code for proteins with apparent molecular weights of 45,000 and 50,000. The A region also includes a 2-kilobase segment involved in regulation of K1 synthesis. The B region encoding five protein species appears responsible for the translocation of the polymer from its site of synthesis on the cytoplasmic membrane to the cell surface. The C region encodes four protein species. Since the three gene clusters appear to be coordinately regulated. we propose that they constitute a kps regulon.     

Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12.

The rfa locus of Escherichia coli K-12 includes a block of about 10 closely spaced genes transcribed in the same direction which are involved in synthesis and modification of the hexose region of the lipopolysaccharide core. We have sequenced the first three genes in this block. The function of the first of these genes is unknown, but we have designated it rfaQ on the basis of its location and similarity to other rfa genes. Complementation of Salmonella typhimurium rfa mutants with E. coli rfa restriction fragments indicated that the second and third genes in the block were rfaG and rfaP. The deduced sizes of the RfaQ, RfaG, and RfaP proteins are 36,298, 42,284, and 30,872 Da, respectively, and the proteins are basic and lack extensive hydrophobic domains. RfaQ shares regions of homology with proteins RfaC and RfaF, which are involved in synthesis of the heptose region of the core. Proteins RfaB, RfaG, and RfaK share a region of homology, which suggests that they belong to a second family of Rfa proteins which are thought to be hexose transferases.     

Genetic Determination of Resistance to Acriflavine, Phenethyl Alcohol, and Sodium Dodecyl Sulfate in Escherichia coli

Wild-type strains of Escherichia coli K-12 are resistant to acriflavine. Gene acrA(+) which determines resistance to acriflavine is located near the lac region of the chromosome. This gene determines not only resistance to basic dyes but also resistance to phenethyl alcohol. Acriflavine resistance was transmitted, together with phenethyl alcohol resistance, from a resistant Hfr strain to a sensitive recipient by mating. Reversion of the mutant gene acrA1 (phenotypically acriflavine-sensitive) to acriflavine resistance was accompanied by a change from phenethyl alcohol sensitivity to resistance, and conversely the revertants selected for phenethyl alcohol resistance were resistant to acriflavine. A suppressor mutation, sup-100, closely linked to the acr locus, suppresses the acrA1 gene (phenotypically acriflavine-resistant), but does not determine resistance to phenethyl alcohol and basic dyes other than acriflavine. The genetic change in the locus acrA1 to types resistant to basic dyes and phenethyl alcohol was accompanied by an increase in resistance to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that gene acrA determines synthesis of a membrane substance. The system seemed to be affected strongly by the presence of inorganic phosphate.     

A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus

The Myxococcus xanthus asg genes ( asgA, asgB , and asgC ) are necessary for production of extracellular A-signal, which is thought to function as a cell-density signal. Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions. In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG , and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon. Surprisingly, asgC767 was found to be a mutant allele of rpoD , the gene encoding the major sigma factor of M. xanthus . The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors. Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes. We found that A-signal restores developmental expression of an A-signal-dependent gene (Ω4521) in the asgC767 ( rpoDEK598 ) mutant background in a manner similar to that seen in the asgA and asgB mutants. Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A-signal production.     

Use of lipophilic cation-permeable mutants for measurement of transmembrane electrical potential in metabolizing cells of Escherichia coli.

Some lipopolysaccharide-defective mutants of Escherichia coli showed, without ethylenediaminetetraacetic acid treatment, a quick and high uptake of lipophilic cations such as triphenylmethylphosphonium and tetraphenylphosphonium. The rate and amount of uptake were comparable to those of an ethylenediaminetetraacetic acid-treated wild type. Transmembrane electrical potential, which was calculated from the distribution of these lipophilic cations between the inside and outside of the mutant cells, was about -150 mV at pH 7.5 and showed a strong dependency on the external pH. One of the E. coli mutants, the acrA mutant, was found to be also permeable to dicyclohexylcarbodiimide, an H+-adenosine triphosphatase inhibitor, and 1-anilino-8-naphthalene sulfonate, a fluorescent dye. The acrA mutant was vigorously motile and highly sensitive to many bacteriophages and colicins. Thus, the acrA mutant is quite useful for the quantitative measurement of transmembrane electrical potential by lipophilic cations in intact and metabolizing cells especially in relation to motility and actions of colicins and bacteriophages.     

Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium.

Analysis of the sequence of a 4.3-kb region downstream of rfaJ revealed four genes. The first two of these, which encode proteins of 27,441 and 32,890 Da, were identified as rfaY and rfaZ by homology of the derived protein sequences of their products to the products of similar genes of Salmonella typhimurium. The amino acid sequences of proteins RfaY and RfaZ showed, respectively, 70 and 72% identity. Genes 3 and 4 were identified as rfaK and rfaL on the basis of size and position, but the derived amino acid sequences of the products of these genes showed very little similarity (about 12% identity) between Escherichia coli K-12 and S. typhimurium. The next gene in the cluster, rfaC, encodes a product which also shows strong protein sequence homology between E. coli K-12 and S. typhimurium, as do the rfaF and rfaD genes which lie beyond it. Thus, the rfa gene cluster appears to consist of two blocks of genes which are conserved flanking a central region of two genes which are not conserved between these species. Although the RfaL protein sequence is not conserved, hydropathy plots of the two RfaL species are nearly identical and indicate that this is a typical integral membrane protein with 10 or more potential transmembrane domains. We noted the similarity of the structure of the rfa gene cluster to that of the rfb gene cluster, which has now been sequenced in several Salmonella serovars. The rfb cluster also contains a gene which lies within a central nonconserved region and encodes an integral membrane protein similar to protein RfaL. We speculate that protein RfaL may interact in a strain- or species-specific way with one or more Rfb proteins in the expression of surface O antigen.     

Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.     

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2.

Two pathways exist for cleavage of the carbon-phosphorus (C-P) bond of phosphonates, the C-P lyase and the phosphonatase pathways. It was previously demonstrated that Escherichia coli carries genes (named phn) only for the C-P lyase pathway and that Enterobacter aerogenes carries genes for both pathways (K.-S. Lee, W. W. Metcalf, and B. L. Wanner, J. Bacteriol. 174:2501-2510, 1992). In contrast, here it is shown that Salmonella typhimurium LT2 carries genes only for the phosphonatase pathway. Genes for the S. typhimurium phosphonatase pathway were cloned by complementation of E. coli delta phn mutants. Genes for these pathways were proven not to be homologous and to lie in different chromosomal regions. The S. typhimurium phn locus lies near 10 min; the E. coli phn locus lies near 93 min. The S. typhimurium phn gene cluster is about 7.2 kb in length and, on the basis of gene fusion analysis, appears to consist of two (or more) genes or operons that are divergently transcribed. Like that of the E. coli phn locus, the expression of the S. typhimurium phn locus is activated under conditions of Pi limitation and is subject to Pho regulon control. This was shown both by complementation of the appropriate E. coli mutants and by the construction of S. typhimurium mutants with lesions in the phoB and pst loci, which are required for activation and inhibition of Pho regulon gene expression, respectively. Complementation studies indicate that the S. typhimurium phn locus probably includes genes both for phosphonate transport and for catalysis of C-P bond cleavage.     

Transformation mapping of the genes controlling tryptophan biosynthesis in Bacillus subtilis

Forty tryptophan auxotrophs of Bacillus subtilis have been placed in six phenotypic classes on the basis of growth responses, accumulation properties, and, in some cases, specific enzymatic defects. Three-point transformation crosses between representative mutants of the six different types have permitted the determination of the orders of the gene loci. In addition, mutational site orders for mutants within each of the classes have been determined by the same techniques. The organization of the cluster of genes controlling tryptophan biosynthesis in B. subtilis appears to be essentially analogous to that of Escherichia coli and Salmonella typhimurium.     

Two classes of region III flagellar genes in Escherichia coli

We infected various nonflagellated mutants of Escherichia coli with fla-transducing phages and followed the kinetics of the appearance of motility. Our analysis revealed two distinct classes of region III fla genes. Class II fla genes (hag, flaD) functioned 15 min later than class I fla genes (flaN, flaB, flaC, flaO, flaA, flbD, flaQ, flaP) in flagellar morphogenesis. We suggest that the two classes of fla genes are involved in two different stages, initiation (class I) and completion (class II), of flagellar formation.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.